Designer protein assemblies with tunable phase diagrams in living cells.

Protein self-organization is a hallmark of biological systems. Although the physicochemical principles governing protein-protein interactions have long been known, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remain challenging to characterize. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies. We devise a new strategy to map high-resolution phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Finally, we find that cotranslational protein binding suffices to recruit a messenger RNA to the designed micron-scale structures.

Evolthon: A community endeavor to evolve lab evolution.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation.

YeastRGB: comparing the abundance and localization of yeast proteins across cells and libraries.

The ability to measure the abundance and visualize the localization of proteins across the yeast proteome has stimulated hypotheses on gene function and fueled discoveries. While the classic C' tagged GFP yeast library has been the only resource for over a decade, the recent development of the SWAT technology has led to the creation of multiple novel yeast libraries where new-generation fluorescent reporters are fused at the N' and C' of open reading frames. Efficient access to these data requires a user interface to visualize and compare protein abundance, localization and co-localization across cells, strains, and libraries. YeastRGB (www.yeastRGB.org) was designed to address such a need, through a user-friendly interface that maximizes informative content. It employs a compact display where cells are cropped and tiled together into a 'cell-grid.' This representation enables viewing dozens of cells for a particular strain within a display unit, and up to 30 display units can be arrayed on a standard high-definition screen. Additionally, the display unit allows users to control zoom-level and overlay of images acquired using different color channels. Thus, YeastRGB makes comparing abundance and localization efficient, across thousands of cells from different strains and libraries.

Synthetic condensate size correlates with yeast replicative cell age.

Yeast divides asymmetrically, with an aging mother cell and a 'rejuvenated' daughter cell, and serves as a model organism for studying aging. At the same time, determining the age of yeast cells is technically challenging, requiring complex experimental setups or genetic strategies. We developed a synthetic system composed of two interacting oligomers, which forms condensates in living yeast cells. Here, we report that these synthetic condensates' size correlates with yeast replicative age, making these condensates age reporters for this model organism.

Protein recognition by a pattern-generating fluorescent molecular probe.

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.